RU2270856C2 - Nutrient medium for submerged culturing plague microorganism of vaccine antibiotic-resistant strain eb p2 - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes the nutrient medium comprising enzymatic hydrolyzate of defatted cow milk proteins as a nutrient base with the content of amine nitrogen 120 mg% and concentration of hydrogen ions (pH value) 7.0 ± 0.2 in the following ratio of components, g/l: enzymatic hydrolyzate of defatted cow milk proteins, 0.3; potassium dihydrogen phosphate, 1.4; potassium hydrogen phosphate, 2.7; magnesium sulfate, 1.1; sodium chloride, 0.1, and distilled water, up to 1 l. Invention provides increasing yield of plague microorganism biomass of the vaccine antibiotic-resistant strain EB P2 in submerged culturing in liquid nutrient medium based on balanced composition of amino acids, peptides, carbohydrates and mineral salts.

EFFECT: improved and valuable properties of nutrient medium, increased yield of biomass.

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Description

The invention relates to the field of microbiology, namely to the development and creation of nutrient media for deep cultivation of the plague microbe of the vaccine antibiotic-resistant strain EB P2.

For deep cultivation of the plague microbe, liquid nutrient media are used based on the enzymatic hydrolyzate of cattle meat (Production Schedule (RP) No. 1386-03 "Plague Live Dry Vaccine" approved by the head of the Scientific Research Institute of the Ministry of Defense of the Russian Federation on 10.06.2003), casein enzymatic hydrolyzate ( RP No. 1386-03 "Plague live dry vaccine", approved by the head of the NIIM MO RF on 10.06.2003), casein acid hydrolyzate (RP No. 1386-03 "Plague living dry vaccine", approved by the head of the NIIM MO RF 10.06.2003 ), yeast extract (Sheremet O.V. et al. ZhMEI - 1987, No. 6, p. 18-21).

The liquid nutrient medium based on the enzymatic hydrolyzate of cattle meat - FGM cattle (g · l ^-1 ):

Khottinger digest - 150.0;

NaCl - 5.0;

KCl - 0.2;

Na ₂ HPO ₄ - 1.0;

the content of amine nitrogen in the medium is 120 ... 130 mg%;

the concentration of hydrogen ions is 7.15 units. pH

Liquid nutrient medium based on enzymatic casein hydrolyzate - FGK (g · l ^-1 ):

FGC - 150.0;

KH ₂ PO ₄ - 1.7;

K ₂ HPO ₄ 2.2;

MgSO ₄ 0.6;

Na ₂ S ₂ O ₃ - 0.6;

the content of amine nitrogen in the medium is 138 mg%;

the concentration of hydrogen ions - 7.2 units pH

The disadvantages of these media include the low level of results on the accumulation of biomass of the plague microbe of the vaccine antibiotic-resistant strain EB P2.

In addition, these media are difficult to manufacture and use scarce and expensive raw materials to produce them.

A nutrient medium is also known for the accumulation of plague microbe biomass at 26 ... 28 ° C based on a yeast extract. The composition of this medium includes a nutrient base, casein hydrolyzate or meat hydrolyzate according to Hottinger or sunflower meal hydrolyzate, sodium chloride and sodium sulfite in different proportions.

The disadvantages of this nutrient medium include: low growth properties, the mandatory preparation of two nutrient bases.

The closest is a nutrient medium based on casein acid hydrolyzate (KGC), successfully used for deep cultivation of the plague microbe of the EB vaccine strain in the technology for preparing the plague live dry vaccine.

Liquid nutrient medium based on acid hydrolyzate of casein KGC (g · l ^-1 ):

KGC - 150.0;

KH ₂ PO ₄ - 2.2;

K ₂ HPO ₄ - 1.7;

MgSO ₄ 0.6;

Na ₂ S ₂ O ₃ - 0.6;

the content of amine nitrogen in the medium is 130 ... 160 mg%;

the concentration of hydrogen ions is 7.15 units. pH

A common with the claimed solution is the presence of potassium salts in these media: potassium phosphoric acid disubstituted, potassium phosphoric acid monosubstituted; magnesium sulfate, sodium chloride.

The main disadvantage of this environment is the low biomass yield of the plague microbe of the vaccine antibiotic-resistant strain EB P2.

The objective of the invention is to increase the biomass yield of the plague microbe of the vaccine antibiotic-resistant strain EB P2 during deep cultivation in a liquid nutrient medium.

This goal is achieved due to the fact that the proposed recipe for the nutrient medium contains a fundamentally new nutrient base: enzymatic hydrolyzate of skim milk cow proteins (FGBOKM) with an amine nitrogen content of 120 mg% in the following ratio of components (g · l ^-1 ):

FGBOKM - 0.3;

potassium phosphoric acid monosubstituted - 1.4;

potassium phosphoric disubstituted - 2.7;

magnesium sulfate - 1.1;

sodium chloride - 0.1;

distilled water - up to 1 liter;

concentration of hydrogen ions 7.0 ± 0.2 units pH

A 1.5-fold increase in biomass yield is due to the presence in the nutrient base of a balanced composition of amino acids, peptides, carbohydrates and mineral salts.

The possibility of carrying out the claimed invention is shown by the following example.

Preparation of a liquid nutrient medium. In a digester with 65 liters of distilled water, 15 liters of enzymatic hydrolyzate of skimmed cow's milk proteins are poured, heated to boiling, and a sample is taken to control and adjust the amount of amine nitrogen.

Salt is introduced into the broth at the rate of: sodium chloride - 0.1; magnesium sulfate - 1.1; potassium phosphoric disubstituted - 2.7; potassium phosphoric acid monosubstituted - 1.4. The mixture is stirred until the salts are completely dissolved, boiled for 5 ... 10 minutes and a sample is taken to control and adjust the pH value. If necessary, the pH value is adjusted with HCl and NaOH solutions within 7.0 ± 0.2 units. pH

The hot medium is filtered through a cotton-gauze filter and Packed in a 20 liter bottle of 10 liter medium. The bottles are sterilized in an autoclave at a temperature of 120 ... 125 ° C for 30 ... 35 minutes.

Thus, the implementation of the invention allows to increase the biomass yield of the plague microbe of the vaccine antibiotic-resistant strain EB P2.

n=10)Table 1 - a comparative characteristic of the biomass yield of the plague microbe of strain EB P2 during deep cultivation in a liquid nutrient medium on devices with a volume of 0.007 m ³ (

n = 10)

Test strain The concentration of living microbial cells (x ± I ₉₅ ) when cultured on a medium ... FGM FGK KGC FGBOKM Y.pestis EB P2 8.0 ± 0.4 7.8 ± 0.2 8.25 ± 0.1 12.2 ± 0.2

Claims (1)

Nutrient medium for deep cultivation of the plague microbe of the vaccine antibiotic-resistant strain EB P2, containing a nutrient base - an enzymatic hydrolyzate of casein skim milk proteins with an amine nitrogen content of 120 mg%, a concentration of hydrogen ions of 6.8 ... 7.2 units. pH, potassium phosphate monosubstituted, potassium phosphate disubstituted, magnesium sulfate, sodium chloride, distilled water in the following ratio of components, g / l: Enzymatic protein hydrolyzate casein skim milk with an amine nitrogen content of 120 mg%, the concentration of hydrogen ions 6.8 ... 7.2 units. 0.3 Potassium phosphate monosubstituted 1.4 Potassium Disubstituted Potassium Phosphate 2.7 Magnesium sulfate 1,1 Sodium chloride 0.1 Distilled water Rest